flow cytometry reinvented

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1 FLOW CYTOMETRY flow cytometry reinvented The Attune Acoustic Focusing Cytometer Precision and at all speeds for rare events and precious samples

2 The Attune Acoustic Focusing Cytometer Precise high collection rates without compromising CVs Fast rare-event detection with dramatically shorter acquisition times Simple no-lyse, no-wash method eliminates cell loss With the Attune Acoustic Focusing Cytometer, you get both high sample input rate and high precision when analyzing precious samples or detecting rare events. You control your sample concentration, flow rate, the number of photons you detect, the length of your experiment, and more. You get the most Hydrodynamic out of your cells with dependable results. But best of all, the Attune makes it easy. focusing 60 instrument 40 Precision and at all sample throughput rates The Attune Acoustic Focusing Cytometer enables higher when you need it most. You ll be able to maintain precise alignment, even at high sample rates of up to 1 ml/min. And because the Attune 20 is not limited by physical 66.8% flow constraints, you can control the amount of time cells spend in the lasers path. This means you can use a slower rate to collect more photons when analyzing dim signals, translating to better clarity of your results and better separation (Figures 1 and 2) PE-A Hydrodynamic focusing instrument % Attune, normal mode % of Max Note: increased precision as seen by the tighter populations % PE-A BL2-A:BL2-A Figure Attune 1. Greater 0 peak separation with phosphospecific Note: antibody staining. Intracellular target detection can be challenging. Jurkat T cells were, stimulated with anti-cd3 and anti-cd28 antibodies, increased then fixed and permeabilized before staining with a PE-labeled phosphospecific antibody. normal The sample was 80run on the Attune precision and as a hydrodynamic focusing and the data compared. The plots show an overlay of the data from stimulated and unstimulated seen by samples the from each system. The data obtained with the Attune Acoustic Focusing tighter Cytometer mode have higher precision 60 (narrower peaks), resulting in greater separation and easier data interpretation. populations. % of Max % BL2-A:BL2-A

3 Conventional Conventional flow Conventional flow flow Attune Attune Attune Attune Attune Attune Conventional Conventional flow Conventional flow flow (Highest (Highest (Highest Attune Attune Attune Attune Attune Attune (Standard (Standard (Standard (High (High (High PE-Cy PE-Cy PE-Cy T T T T 1 4 T 6 4 T PE-Cy PE-Cy PE-Cy PE-Cy 3 21 PE-Cy PE-Cy (Highest (Highest (Highest (Standard (Standard (Standard (High (High (High Figure 2. Sensitivity through control of cell transit times. Fluorescent microspheres (Spherotech Rainbow 3.2 μm) were run on a high-end conventional flow and on the Attune Acoustic Focusing Cytometer using a 488 nm blue laser (top row) and a 40 nm violet laser excitation (bottom row). The conventional flow was run at its highest setting, at a sample input rate of 1 μl/min (left column). The Attune was run at 2 different settings: at its standard setting, at a 0 μl/min sample input rate (middle column), and at its highest setting, with a 0 μl/min sample input rate and 4x increase in the amount of time the particles are illuminated by the laser (right column). The Attune standard settings resulted in equal to or better than the conventional flow, while the high of the Attune instrument resulted in detection of the faintest beads not detected by the conventional flow. Rare events detection times faster The Attune Acoustic Focusing Cytometer achieves sample throughput at rates over times faster than other s up to 1,000 µl per minute, enabling rapid detection of rare events with reliable accuracy while aborting no data (Table 1, Figure 3). The Attune is designed to collect up to 20 million events per run and has adjustable collection rates of 2 1,000 µl/min. CD4R/B220-PE fluorescence Gated on live CD19 cells at collection rate of 00 ul/min Table 1. Comparison of acquisition times with a hydrodynamic focusing or the Attune Acoustic Focusing Cytometer, using a blood sample from an aplastic anemia individual, each with a stop gate set on 1 million granulocyte events. Instrument (collection rate) 6 Time to acquire Relative rate 1 million compared to hydrogranulocyte events dynamic focusing Hydrodynamic focusing (high) 63 min 33 sec 4 Attune Acoustic Focusing Cytometer (200 µl/min) 13 min 20 sec 4.8x faster 3 Attune Acoustic Focusing Cytometer (00 µl/min) min 47 sec 11.0x faster Attune Acoustic Focusing Cytometer (1,000 µl/min) 3 min 13 sec 19.7x faster CD317-Alexa Fluor 488 fluorescence Figure 3. Collecting more than 1 million live cells and detecting a rare population of dendritic cells of 0.2% with mouse splenocytes. Plasmacytoid dendritic cells (pdcs) are a specialized cell population that produces large amounts of type I interferons in response to viruses and are identified using the immunophenotype CD19, B220high, CD317+. Four-color staining of mouse splenocytes included CD19 Blue, CD317 Alexa Fluor 488, CD4R/B220-PE direct conjugates, and SYTOX AADvanced Dead Cell Stain. A gate was made on live cells using SYTOX AADvanced Dead Cell Stain, followed by gating on CD19 cells. A two-parameter plot of CD4R/B220 vs. CD317 was used to identify pdcs. A collection rate of 00 µl/min was used to acquire 1.3 million total cells with a cell concentration of 7.x^7 cells/ml. Plasmacytoid dendritic cells were identified as dual B220 +/CD317+ (upper right quadrant) and constitute 0.81% of live CD19 cells, which is 0.194% of total splenocytes.

4 SSC Attune sample rate: 1,000 µl/min CD CD34 Figure 4. Identification of CD34 + cells from peripheral blood. Peripheral blood from a normal donor was stained and run on the Attune Acoustic Focusing Cytometer at a collection rate of 1,000 μl/min with a stop gate set at 00,000 total cells. A rare population of 0.04% CD34 + cells (red box) was identified within the population of cells with an acquisition time of 4 minutes, 28 seconds. In addition, the Attune provides direct cell counts without the need for expensive counting beads with volumetric analysis (Figure 4, Table 2). Table 2. Comparison of absolute cell counting with a hydrodynamic focusing or the Attune Acoustic Focusing Cytometer. Instrument Sample Flow Rate (µl/ min) Acquisition Time Percentage of CD34 + Cells CD34 + Cells/µL, Direct Measurement Attune Acoustic Focusing Cytometer 1,000 4 min, 28 sec 0.0% 0.0 High-end conventional min, 46 sec 0.02% N/A Dilute your samples, not your data quality Washing and lysis of red blood cells (RBCs) can cause significant cell loss and damage [1]. Significantly higher sample collection rates allow the Attune to deliver a no-wash, no-lyse protocol to minimize cell loss and simplify sample preparation (Figure ). This feature is particularly useful for samples that are inherently low in concentration. Dilute samples like cerebrospinal fluid (CSF), stem cells, and any sample with low cell numbers can take a long time to acquire. With the Attune, even dilute samples can be acquired quickly without compromising data. Difficult-to-collect samples like mouse blood and bone marrow, thin-needle aspirates, or any sample with low cell yield can be stained and then diluted without washing or performing RBC lysis. High rate collection makes acquisition possible you can run up to 4 ml in just four minutes. No sample loss occurs from sample preparation, and full panel testing is possible for all precious samples. Figure. Eliminating sample preparation without compromising data. Normal whole blood (0 µl) was labeled with CD4 Blue, CD3 Alexa Fluor 488, and CD4 PerCP-Cy. direct conjugates. After 1 minutes of incubation, µl of the stained whole blood was diluted into 4 ml PBS, and data were acquired on the Attune without RBC lysis or washing. A fluorescence threshold was set on the Blue dye to include only CD4-positive cells. A gate was created around the lymphocyte population in a CD4 vs. SSC plot (A) to analyze the mutually exclusive CD4 and CD8 populations (B). By diluting the sample and using a rate of 00 µl/min, all sample preparation steps can be eliminated without compromising the data.

5 Cell cycle analysis using the Attune Acoustic Focusing Cytometer Cell cycle analysis is just one example of where it is critical to precisely detect differences in fluorescence intensity between multiple cell populations. With the Attune Acoustic Focusing Cytometer, minimal variation in results is seen regardless of sample throughput rate. You no longer need to sacrifice throughput for (Figure 6). Minimal variation, even at high sample rates (Figures 6 and 7) Less variability in results (see changes in %S phase from Table 1) No sacrifice of for speed %CV C-Low (12 µl/min) C-Med (3 µl/min) C-High (60 µl/min) Attune (2 µl/min) Attune (0 µl/min) 60 µl/min G O /G 1 CV 1,000 µl/min Attune (200 µl/min) Attune (00 µl/min) Attune (1,000 µl/min) Figure 7. Percent CV of G 0 / G 1 at different flow rates on the Attune and a competitor (C) at different sample rates. Note the minimal change in variability (%CV) for the Attune Acoustic Focusing Cytometer, even at a high sample rate. A High-End Hydrodynamic Focusing Cytometer Low - 12 µl/min B Attune Acoustic Focusing Cytometer 2 µl/min Five other cell types were compared to a high-end hydrodynamic focusing, including ModFit analysis in each case. The sample throughput rate of 2uL/min was used for the analysis (Figure 8). C Medium - 3 µl/min D 200 µl/min Ramos B Cells A Hydrodynamic System G0G1 CV = 3.3% B Attune Acoustic G0G1 CV = 3.11% E High - 60 µl/min F 1,000 µl/min KG-1 a Myeloblast Cells C Hydrodynamic System G0G1 CV =.3% D Attune Acoustic G0G1 CV = 3.8% HL60 Promyeloblast Cells E Hydrodynamic System G0G1 CV = 6.% F Attune Acoustic G0G1 CV = 2.14% Figure 6. Minimal data variation at high sample rates with the Attune Acoustic Focusing Cytometer. Jurkat cells were fixed and stained with propidium iodide, treated with RNase, and analyzed at a concentration of 1 x 6 cells/ml on a high-end instrument that uses hydrodynamic focusing, and on the Attune Acoustic Focusing Cytometer at different sample rates. The left peak in all graphs reflects cells in G 0 /G 1 phase, while the right peak reflects cells in G 2 /M phase. Note that as sample rates increase on the instrument that uses hydrodynamic focusing, the width of the G 0 / G 1 and G 2 /M peaks increase, whereas for the Attune the peaks are relatively stable, even at the highest sample rate of 1,000 µl/min. U266 Myeloma Cells G Hydrodynamic System H Attune Acoustic G0G1 CV = 7.17% G0G1 CV = 4.41% ST486 B Cells I Hydrodynamic System J Attune Acoustic G0G1 CV =.84% G0G1 CV = 2.81% Figure 8. Attune Acoustic Focusing Cytometer exhibits greater precision. Data collected on the Attune exhibits lower coefficients of variation (%CV) and more distinct cell populations when compared to a high-end hydrodynamic focusing flow.

6 What is acoustic focusing cytometry? The Attune Acoustic Focusing Cytometer is the first that uses ultrasonic waves (over 2 MHz, similar to those used in medical imaging), rather than hydrodynamic forces, to position cells into a single focused line along the central axis of a capillary (Figure 9). Acoustic focusing is largely independent of the sample input rate, enabling cells to be tightly focused at the point of laser interrogation regardless of the sample-to- ratio (Figure ). This, in turn, allows slowed cell velocity to collect more photons for high-precision analysis at unprecedented volumetric sample throughput. The Attune accomplishes all this without high velocity or high volumetric fluid, which can damage cells. In addition, volumetric syringe pumps enable absolute cell counting without beads minimizing cost and sample preparation time. In contrast, s that use hydrodynamic focusing maintain the same sample speed at all flow rates, causing cells to lose focus as the sample core widens to increase flow rate. A. Acoustic focusing = better precision Acoustically focused sample stream Laser (cross-section) Acoustic focusing = OFF Sample is unfocused Acoustic focusing = ON Sample is focused Figure 9. Acoustic focusing in action. Fluorescent microspheres were applied to the capillary system of an acoustic focusing. Beads flow through randomly without any acoustic focusing (left). With the application of acoustic focusing, the beads are focused into a single line (right). No more tradeoffs Hydrodynamic focusing s have many limitations. As a result, users often have to sacrifice: Throughput for Features for ease of use Performance for price Power for footprint B. Traditional hydrodynamic focusing: compromised of data quality Hydrodynamic core device Laser (cross-section) Acoustic focusing technology keeps cells within a confined focal point, so these tradeoffs are not required. Figure. Acoustic focusing vs. traditional hydrodynamic focusing. (A) In acoustic focusing, cells remain in tight alignment even at higher sample rates. With this tight alignment, cells pass through the laser beam at its optimal focal point, resulting in less signal variation and improved data quality. (B) In traditional hydrodynamic focusing, increasing the sample rate results in widening of the sample core stream. The speed at which cells pass through the laser is not changed, and is determined by the speed of the fluid flow. Cells are distributed throughout the sample core stream because of reduced differential pressure between sample stream and stream, resulting in reduced cell focusing. Cells are not in tight alignment as they pass through the laser beam, resulting in increased signal variation and compromised data quality.

7 Ideal for both standard and specialized research applications Most standard applications for flow cytometry have been tested on the Attune, including: Live/dead cell discrimination Cell cycle analysis Cell proliferation assays Basic phenotyping (up to 6 colors) Rare-event detection Apoptosis Phagocytosis Detection of phosphoproteinsphagocytosis Other intracellular markers Many standard cell types have been tested, including: Jurkat cells HL60 promyoblast cells U266 myeloma cells, Mouse splenocytes Mouse blood HeLa human cervical carcinoma cells Bovine pulmonary artery epithelial (BPAE) cells 3T3 mouse embryo fibroblast cells Chinese hamster ovary (CHO) cells Human mesenchymal stem cells (hmsc) E. coli, S. aureus Human platelets Human whole blood Data have also been collected in specialized research areas such as oceanography and microbiology. Cells up to 0 μm in diameter have been run without clogging our 200 μm flow cell, with a minimum particle size specification of 1 μm; however, researchers are constantly pushing these limits with the Attune. Service and support The Attune Acoustic Focusing Cytometer is backed by Life Technologies worldwide technical support and service programs. We are focused on delivering personalized service from the time our sales representative walks through the door and throughout the life of your Attune instrument. The Attune is fully supported for one year with our extensive service plan, which includes: Comprehensive training Application and assay support Worldwide technical service Preventive maintenance Extended service plans are also available. For more information about available service plans, go to 1. Gratama JW, Menéndez P, Kraan J, Orfao A (2000) Loss of CD34(+) hematopoietic progenitor cells due to washing can be reduced by the use of fixative-free erythrocyte lysing reagents. J Immunol Methods 239:13 23.

8 Attune Acoustic Focusing Cytometer Software that performs to your specifications User-defined analysis, flexible setup Attune Cytometric Software is designed to provide powerful user-defined analysis using an intuitive interface for simplified experimental analysis (Figure 11). Templates can be built around specific applications and saved for consistent experimental design. Compensation is automated or user-defined and can be set up using a compensation guide. Utilities such as quick-save, drag-and-drop, and copy-and-paste provide rapid manipulation with commonly used functions. Experiments can be easily set up with automated settings that can be completely customized and saved for future experiments. No software licensing fees The Attune Cytometric Software can be downloaded without licensing fees. The software can be added to any desktop or laptop computer at your institution without any additional costs. Results can easily be analyzed at your convenience at your own computer, allowing the next user to run experiments on the Attune. Instrument controls Instrument status Control sample flow rate Select level Run startup/shutdown protocol System performance tracking by date Visualization tools Plot overlay or side-by-side graph comparison Standard or custom gate shapes Histograms, dot plots, density plots Customize statistics Copy and paste plots into reports (Microsoft Word or PowerPoint software) Analysis setup Save protocol templates Automated or user-defined compensation Raw data capture Data display in linear, log, or lin/lg scales Set universal compensation across multiple data sets File format compatibility Save data and images in PDF format for reports Data format in standard FCS 3.0 for compatibility with third-party analysis tools (i.e., FlowJo, ModFit LT, FCS Express software programs) Statistics in.csv format

9 Instrument performance tracking The Attune Cytometric Software provides automated baseline calculation and performance test functions with minimal user interactions. Performance tracking is facilitated through the reports function in the software. Instrument performance tracking is critical for collecting and analyzing accurate experimental data. The Attune Performance Tracking Beads are designed for use with the Attune Cytometric Software to automatically characterize, track, and report performance measurements of the Attune Acoustic Focusing Cytometer (Figure 12). The beads are used to define a baseline and conduct daily measurements of the. Vials contain enough beads for 0 measurements, and specific information for each lot is downloaded to the Attune Cytometric Software prior to use. Performance tracking for the Attune includes a comprehensive set of procedures to monitor the daily performance of the instrument. The performance tracking process involves: Running performance tracking beads Monitoring changes in the coefficient of variation and in the associated PMT voltage Tracking linearity of instrument performance Evaluating the detector and background over time Automatic setting of laser delay Instrument performance is affected by the lasers, optics, and fluidics of the instrument. For optimal instrument performance, it is critical to follow the manufacturer s recommended schedules for maintenance. Figure 11. Example of Attune Cytometric Software. The instrument control panel on the left side of the screen is used for experiment setup and control of laser selection, optics, and fluidics (flow rates). The main frame of this screen shows data analysis plots, while the right panel shows sub-file organization. Figure 12. Example of Levey-Jennings plot showing Attune performance over time. This report is generated using data from performance tracking beads.

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12 Ordering information Description Quantity Part number Attune Acoustic Focusing Cytometer (includes computer, monitor, startup solutions, installation, and warranty) Attune Focusing Fluids, 1X solution, 1 L Attune Focusing Fluids, X solution, 1 L Attune Focusing Fluids, 1X solution, 6 x 1 L Attune Wash Solution Attune X Shutdown Solution Attune Performance Tracking Beads ( x 6 beads/ml) Life Technologies offers a breadth of products DNA RNA PROTEIN CELL CULTURE INSTRUMENTS For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. LIMITED USE LABEL LICENSE: No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 791 Van Allen Way, Carlsbad, California Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Printed in the USA. CO